Over 200,000 Americans will require ACL reconstructive surgery in 2001 with a price tag exceeding 5 billion dollars. The ACL serves as a primary stabilizer of the knee and is most often susceptible to rupture or tear resulting from a flexion-rotation-valgus force associated with sports injuries. Tissue engineering can potentially provide improved clinical options in orthopaedic medicine through the generation of biologically based functional tissues in vitro for transplantation at the time of injury or disease. A tissue engineered ACL with the appropriate biological and mechanical properties available at the time of reconstruction would eliminate the limitations associated with present day surgical techniques, i.e., eliminate the need for autologous patellar or hamstring tendon harvest and/or provide a unlimited source of allogenic ligaments. The elimination of the major debilitating side effect of current treatment--donor-site morbidity--would drastically improve patient outcomes. The overall goal of this project is to engineer viable autologous tissue engineered ACLs eliminating the need for current autologous hamstring or patellar tendon harvest. The final outcome of Phase II will be an engineered ligament ready for human clinical trials. Tissue Regeneration Inc., (TRI) preliminary studies and Phase I results demonstrate the potential of an innovative tissue engineered ligament to serve as a viable replacement for torn or ruptured ACLs in vivo. Specifically, a novel silk fiber-based matrix has been identified, designed and developed, and when seeded with human bone marrow stem cells (BMSCs), supports the specific development of ligament tissue. This tissue expresses biocompatible, biochemical and biomechanical relevant traits to be expected for an ACL. Based on these Phase I studies, we propose to further explore the relationship between a rigorous in vivo intra-articular environment and resulting ACL tissue development (both histomorphologically and mechanically). This study will elucidate key structure-function relationships between initial engineered ACL properties (e.g., matrix linear stiffness) and the in vivo intra-articular remodeling cascade. The potential to restore ACL and knee joint functionality will be determined and assessed in relation to established Phase II milestones in order to reach a "go/no go" for Phase III (human clinical trials) and eventual commercial markets.